Although every cell in our body contains the same genetic information, the differential regulation of those genes has dramatically different outcomes. This proposal aims to study how CTCF organizes chromatin in both space and time to establish a cell's genomic and epigenomic program during differentiation. Loss of a single copy of CTCF leads to aggressive tumor formation, with high levels of invasion and metastasis. Our current understanding of how CTCF maintains a stable genomic program and regulates cellular differentiation is based on data rich genome-wide mapping; however, integration of this data is hindered by our current knowledge gap with respect to the biochemical properties of CTCF. This proposal aims to fill this knowledge gap in the following ways. First, we will characterize the structural and biochemical properties of CTCF using single-molecule methods and four-dimensional live cell imaging to test whether CTCF is necessary and sufficient to form and maintain chromatin loops in vitro and in live cells. Second, we aim to map how epigenetic changes to both chromatin and to CTCF regulate DNA-binding stability using a high-throughput, DNA-binding assay. Because this assay is capable of directly measuring affinities across tens to hundreds of thousands of DNA sequences, we can computationally correlate sequence affinities to ChIP-Seq datasets to model dynamic changes in CTCF-mediated folding of the genome. Finally, ncRNAs have been recently shown to influence chromatin topology both in coordination with and antagonistic to--CTCF, to regulate pleiotropic biological processes including embryogenesis, limb development, X-chromosome inactivation, and VDJ recombination4. We aim to identify those CTCF- bound ncRNAs using RIP-seq (chromatin immunoprecipitation followed by reverse transcription and sequencing). We further aim to develop a topology-dependent RNA-chromatin conformation capture method that will map non-coding RNAs to their association sites across the 3D genome using proximity ligation of RNA and DNA molecules. Like Hi-C, which maps DNA-DNA contacts across the 3-D genome, this approach is conceptually simple but has the potential to uncover chromatin association sites for hundreds or thousands of uncharacterized ncRNAs.